Ensuring that our proposed viral diagnostic test not only abides to the theory but is also truly applicable in a real setting, we tested our modified methodology of Rolling Circle Amplification with synthetic viral gene fragments. The specific modifications are outlined in project description and experimental sections.
Team:TAS Taipei/Proof Of Concept
Summary
Padlock Probe Design
For our padlock probe design, we decided to target the Spike (S) glycoprotein gene for SARS-CoV-2 and the Hemagglutinin (HA) gene for Influenza A and B due to their high copy number in viruses. In order to create a highly specific target sequence, the sequence had to be unique to each virus type while also being able to recognize subtle mutations over time and locations. To that end, we aligned the nucleotide sequences of the S gene from several different SARS-CoV-2 variant strains as well as other related coronaviruses (such as SARS and MERS). We identified a 36 bp sequence that was highly conserved between various mutations within the SARS-CoV-2 strains, while also exhibiting significant differences with SARS and MERS (Figure 1). We performed a similar alignment analysis of the HA gene of Influenza A and Influenza B to identify highly specific target sequences for these viruses (Figure 2-3). All sequences were obtained from NCBI GenBank and compiled through BioEdit. Both DNA and RNA synthetic viral targets were synthesized via IDT.
Figure 1: We chose the SARS-CoV-2 target sequence located in the Spike (S) gene as a target based on alignment data of various strains of SARS-CoV-2 (2019nCov), SARS, and MERS. We chose a 36 nucleotide sequence that is perfectly conserved with those of SARS-CoV2 while showing minimal matching with SARS and MERS.
Figure 2: We chose the influenza A target sequence located in the hemagglutinin gene as a target based on alignment data of various strains of influenza A (H1N1pdm09) and influenza B (Victoria Lineage). We chose a 36 nucleotide sequence that is perfectly conserved with those of H1N1 influenza A while not matching those of influenza B.
Figure 3: We chose the influenza B target sequence located in the hemagglutinin gene as a target based on alignment data of various strains of influenza A (H1N1pdm09) and influenza B (Victoria Lineage). We chose a 36 nucleotide sequence that is perfectly conserved with those of H1N1 influenza A while not matching those of influenza B.
To target these viruses, we designed PLP sequences by adding half of the reverse complement of the target sequence to each side of a standard and tested PLP backbone. Our design process allows our probes to target the selected virus accurately, no matter the case, location, and similarity between viral subtypes.
DNA vs RNA Testing
Using our padlock probe designs, we employed our modified version of the Rolling Circle Amplification with our selected enzymes SplintR ligase and Φ29 DNA polymerase.
Figure 16a: The RCA reaction for DNA Target. T7 ligase is used.
Figure 16b:The RCA reaction for RNA Target. SplintR ligase is used in addition to the RNase Inhibitor.
After optimizing our test, we found that we were able to add all RCA components into one tube for the room temperature reaction to take place. For DNA testing, we would add the synthetic viral DNA target, the target specific padlock probe, T4 ligase, deionized water, NaOH, and a ligation solution with DTT, MgCl2, and ATP which serves the hybridization and ligation portion of the test. We would then add the forward primer, reverse primer, phenol red, Φ29 DNA polymerase, and an amplification solution with DTT, MgCl2, (NH4)2SO4, dNTPs, NaOH, and deionized water for the signal amplification and colorimetric readout portion of the test (Figure 4a).
For RNA testing, we needed to replace T4 DNA ligase with SplintR ligase for the ligation of the hybrid of DNA padlock and synthetic viral RNA target. To prevent the degradation of RNA from RNases, we also added in Protector RNase Inhibitor from Sigma (Figure 4b).
Video 1: RCA reaction ran using Influenza A DNA: In tube G, a reaction was run with no target present. In tube H, a reaction was run with 0.0625uM Influenza A DNA target. Since this is a Influenza A test, all padlock probes used were Influenza A padlock probes.
Video 2: RCA reaction ran using SARS-CoV-2 RNA: In tube A, a reaction was run with no target present. In column B, a reaction was run with 0.0625uM SARS RNA target. In tube C, a reaction was run with 0.0625uM SARS-CoV-2 RNA target. Since this is a SARS-CoV-2, all padlock probes used were SARS-CoV-2 padlock probes.
The videos shown above are examples of our RCA tests for DNA and RNA targets, which we recorded as a timelapse. As shown, a color change occurs from purple to yellow when the synthetic viral fragment of the virus tested or targeted is present. Otherwise, the color remains around purple.
Figure 5: RCA test with synthetic viral DNA targets with photos of tubes at 0, 30, 60 and 90 minute time intervals with their respective hue and pH.
Figure 6: RCA test with synthetic viral RNA targets with photos of tubes at 0, 30, 60 and 90 minute time intervals with their respective hue and pH.
In the tables shown above, a more thorough view into the color changes of all RCA tests for each virus are shown. Our results indicate that in both the DNA and RNA RCA tests, a color change from purple to yellow occurs in roughly 60 minutes for Influenza B and SARS-CoV-2 testing, when the tested synthetic viral gene fragment is present. As for Influenza A, it may require more time and the color change usually occurs by 90 minutes if tested with RNA. Using our modeling software tool, we captured the hue of the solution in the timelapses of our reaction and converted them to pH values. We plotted these pH values over time in the graphs below.
Figure 7: pH over Time Graph of Influenza A RCA Test for both synthetic viral DNA and RNA targets.
Figure 8: pH over Time Graph of Influenza B RCA Test for both synthetic viral DNA and RNA targets.
Figure 9: pH over Time Graph of SARS-CoV-2 RCA Test for both synthetic viral DNA and RNA targets.
The colorimetric indicator we chose is phenol red, which tends to be purple at around pH 8.0, orange at around pH 7.2, and yellow at pH 6.8. Analyzing the data from these graphs, it is evident that DNA testing yields a much quicker color change from purple to yellow than RNA testing. However, both are still operable at reasonable time periods, especially considering that all RCA tests were conducted at room temperature. Testing both DNA and RNA viruses are crucial because although the high impact viruses we selected to detect, Influenza A (H1N1pdm09), Influenza B (Victoria Lineage), SARS-CoV-2, are RNA viruses, other notable viruses such as smallpox and papillomaviruses are DNA viruses. Analyzing the rate of our reaction has also provided useful quantitative data to show which RCA conditions present the optimal rate of viral target detection.
Sensitivity Analysis
Another test we conducted concerns the analytical sensitivity of our assay. In these tests, we selected the SARS CoV-2 synthetic viral target and proceeded to conduct serial dilutions to test RCA under different concentrations. Using a micromolar concentration as a standard, we then compared the time of color change that occurred to samples at nanomolar, picomolar, femtomolar, and attomolar concentrations.
The table below indicates the number of RNA strands that correlates with each concentration.
Figure 10: Conversion of RNA Concentration to Number of RNA strands.
Video 3: RCA Sensitivity Test on SARS-CoV-2
In the timelapse of the SARS-CoV-2 RCA test above, tube A has no target, tube B has 0.0625uM target, tube C has 0.00625uM target, tube G has 0.0625nM target, tube D has 0.027pM target, tube E has 0.0027pM target, tube F has 0.00027pM target, and tube H has 0.027fM target, and tube I has 0.027aM target. All targets and padlock probes are SARS-CoV-2. The timelapse shows a color change gradient from the 0.0625uM to 0.027fM target concentrations, while the no target negative control remained purple. Similar to the negative control, the RCA reaction did not change color when 0.027aM concentration of target was added. This suggests that our diagnostic test can detect up to 0.027fM concentrations of synthetic viral RNA.
Figure 11: pH over Time Graph of SARS-CoV-2 RCA Test under different RNA concentrations.
The pH over time graph of the RCA solution in the reaction tubes confirms our observation. We can detect up to 0.027fM concentrations which is equivalent to around 16.26 RNA strands.
Specificity Test
In addition to determining how sensitive our test can be, we also wanted to know the specificity of our assay. This means to us whether differing sequences of synthetic viral targets that reflect different virus strains can be differentiated by our test. To find out, we used no target, a mismatched target, and a matched target for each of our tests as determined by our padlock probe. Each padlock probe has a specific sequence that is only fully complementary to its corresponding target, which should only instigate a reaction.
Figure 12: Different Tests Ran to Determine the Specificity of Our Padlock Probe
The table below shows the different synthetic RNA target and padlock probe combinations used in each of the three virus tests focused on. (+) means the presence of the target while (-) means a lack thereof.
The SARS-CoV-2 and the SARS targets which are both coronaviruses, differ by 18 nucleotides in alignment. The Influenza A and Influenza B targets which are both influenza viruses, differ by 27 nucleotides in alignment.
Video 4: Specificity Test on SARS-CoV-2 RNA RCA
The timelapse of our RCA test above is on SARS-CoV-2, meaning all padlock probes used were SARS-COV-2. In tube A, there is no target. In tube B, there is a SARS RNA target (mismatched target). In tube C, there is a SARS-CoV-2 target (matched target).
Video 5: Specificity Test on Influenza A and B RNA RCA
The timelapse of our RCA test above is on Influenza A and B. Tubes A to C are Influenza A tests, meaning Influenza A padlock probes are used. In tube A, there is no target. In tube B, there is an Influenza B RNA target (mismatched target). In tube C, there is an Influenza A RNA target (matched target). Tubes D to F are Influenza B tests, meaning Influenza B padlock probes are used. In tube D, there is no target. In tube E, there is an Influenza A RNA target (mismatched target). In tube C, there is an Influenza B RNA target (matched target).
Figure 13: pH over Time Graph of Specificity Test on SARS-CoV-2 RCA Test. No target (negative control). 1: SARS target. 2: SARS-CoV-2 target.
Figure 14: pH over Time Graph of Specificity Test on Influenza A RCA Test. No target (negative control). 1: Influenza B target. 2: Influenza A target.
Figure 15: pH over Time Graph of Specificity Test on Influenza B RCA Test. 0: No target (negative control). 1: Influenza A target. 2: Influenza B target.
Our graphs once again confirm that a notable difference in rate of change occurs between the presence of the mismatched viral sequence and the matched viral sequence. This suggests that our diagnostic test has some level of capability in differentiating between the actual viral sequence being targeted and the sequence of a close viral strain that is not being targeted. To set a threshold to indicate when a patient should view their RCA test tube’s color, we can still identify a specific type of virus.
The Bigger Picture
Our sequence designs, RCA colorimetric experiments and pH measurements ultimately serve as the viral diagnosis and software portion of our project. But to truly bring it to life, we have to incorporate portions such as our modeling hardware piece, where we presented a theory and 2 in 1 saliva collection and purification device design to allow nucleic acid isolation. Inputting the pH change of our reactions into our model, we determined the RNA concentration of a reaction. We were then able to identify the viral load of the patient. Although viral load does not correlate with the severity of the viral disease, it can be a good indicator of the time since viral exposure and the level of infectivity of a sick individual. From a Human Practices perspective, this presents ways to seek immediate attention for the benefit of oneself and the others around the individual. This ultimately allows us to implement our project as a viral diagnostic home test kit that is not only highly usable, but provides useful information to an individual being tested.
Figure 16: Flowchart of our Viral Spiral Project for Viral Diagnosis
References
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